Project Summary/Abstract The aggregation of the 37-amino acid polypeptide human Islet Amyloid Polypeptide (hIAPP, amylin), as either insoluble amyloid or as small oligomers, appears to play a direct role in the death of pancreatic ?-islet cells in type 2 diabetes. It is known that several organisms express non-amyloidogenic variants of IAPP (such as rat and mouse) and are not known to develop diabetes naturally. Conversely, several organisms express highly amyloidogenic variants of IAPP (such as human, cat and primates) and are known to develop diabetes. Despite the significant amount of genetic information available, no comprehensive study has been conducted to directly correlate IAPP aggregation potential to the propensity to develop diabetes within the animal kingdom. In this work, we will compare the aggregation potential and cellular toxicity of naturally occurring IAPP variants from organisms known to, or known not to, develop diabetes. We propose to (1) construct and screen a library of IAPP variants genetically fused to enhanced green fluorescent protein. In this screen, the gene for IAPP is genetically fused to the gene for enhanced green fluorescent protein (EGFP). When the IAPP-EGFP fusion protein is expressed in E. coli the natural propensity of IAPP to aggregate precludes EGFP from folding and fluorescing. However, variants that resist aggregation, will allow the EGFP to fold and fluoresce brightly. (2) Investigate the aggregation potential of synthetic IAPP variants. Using Thioflavin T binding, atomic force microscopy, circular dichroism and antibody detection assays, we will quantitate the ability of the IAPP variants to aggregate. (3) Quantitate the toxicity of IAPP variants on mammalian cells. We will incubate the IAPP variants with mammalian cells and determine cell viability using MTT assays. (4) Identify variants capable of inhibiting human IAPP aggregation. We will incubate hIAPP with each synthetic IAPP variant to determine which variants are capable of inhibiting the formation of toxic hIAPP species. We believe these experiments have the potential to (i) further our understanding of the sequence determinants of IAPP aggregation (ii) identify the link between IAPP aggregation and the propensity to develop diabetes and (iii) identify new peptide sequences capable of inhibiting the formation of toxic hIAPP.